A topical microbicide to prevent sexual transmission would contribute substantially to controlling the spread of HIV. RNA interference (RNAi) is an evolutionarily conserved, host defense against viruses and transposable elements, which uses small double-stranded RNAs, called small interfering RNAs (siRNA), to silence gene expression with exquisite specificity. siRNAs targeting viral coreceptors and/or HIV genes can prevent viral entry or suppress HIV replication, even in productively infected cells. By applying siRNAs mixed with a lipid carrier intravaginally in mice, siRNAs are delivered uniformly deep into the vagina and ectocervix and can silence an endogenous gene throughout the mucosa and submucosa. Silencing persists for at least 9 days without apparent toxicity, suggesting that an siRNA-based microbicide might have improved compliance, since it would not need to be used just before exposure. siRNAs targeting viral genes protected mice against intravaginal herpes simplex virus 2 (HSV-2), an important cofactor for HIV transmission. Based on these exciting preliminary data, this Integrated Preclinical/Clinical Program for Topical Microbicides will test the hypothesis that RNAi can form the basis of an anti-HIV microbicide. The siRNA complexes will be refined for improved delivery to cells important in HIV transmission to optimize protection from viral challenge without toxicity. The groundwork for performing a pilot study to test the safety of an siRNA-based microbicide will begin to be laid, but initiating pilot clinical studies is outside the scope of this proposal. Many steps are needed to develop an siRNA-based microbicide. Although mice were protected from vaginal HSV-2 challenge, the cell types targeted by HSV-2 (epithelial cells, neurons) are different than those important for HIV transmission. Moreover, although silencing was seen throughout the mouse genital tissue, some of the cells responsible for HIV transmission are rare in the normal uninflamed genital mucosa. The major first aim of this program is to determine which cells in the mucosa are efficiently targeted by siRNA-lipid complexes and, if necessary, to design alternate strategies to deliver siRNAs into the key cells believed to be important for HIV transmission - Langerhans cells and lamina propria T cells, dendritic cells and macrophages. Another key aim is to optimize the siRNAs for stability and efficiency of intracellular silencing and to develop a strategy for formulating the siRNA compounds into a gel suitable for vaginal application. Modifications will be tested iteratively in vitro in cell lines and primary cells, in human cervicovaginal explants and in mice. Lead candidates will then be tested in the macaque for protection against SHIV challenge.